The role of autoantibodies in diseases is a subject of intense research. Autoantibodies form a diverse repertoire with the capacity to target antibody, peptidic, or nucleic acid self-antigens. These autoantibodies may be monovalent or polyreactive and may exhibit varying affinities and avidities; dissociation constants can range from 10−5 to 10−8 M. While autoantibodies are widely implicated in autoimmune diseases, a subset of autoantibodies also plays a non-pathological role in maintaining immunological homeostasis and a properly functioning immune system. There is mounting evidence that autoantibodies are essential to the survival and/or development of T and B lymphocytes in the peripheral immune system, defense against microbial infections, inflammation suppression, mediation of efferocytosis, anti-idiotypic immunomodulation, and regulation of cytokine production. See, Elkon et al. (2008) Nat Clin Pract Rheumatol 4(9): 491-498; Silverman et al. (2009) Discovery Medicine 8(42): 151-156; and Lacroix-Desmazes S. et al., (1998): J Immunol Methods 216, 117-137.
Detection of autoantibodies in patients suspected of having, or diagnosed with, an autoimmune disease has significant utility in research or in the clinical context. Autoantibodies can serve as a diagnostic marker or indicator of disease progression. For instance, rising levels of dsDNA antibodies in SLE (systemic lupus erythematosus) correlate with increasing disease activity and sometimes precede clinical relapse in patients. Numerous autoantibodies have been linked in the literature to various disorders. See, Watts et al. (2002) Medicine 30(10): 2-6. Some examples include anti-amyloid-β in Alzheimer's disease; anti-thyroglobulin in thyroiditis; anti-tubulin in autoimmune liver disorders, autoimmune hearing loss, and autoimmune thyroid diseases; rheumatoid factors in rheumatoid arthritis; and anti-dsDNA antibody (Ab) in SLE.
Measurement of autoantibodies is also useful for therapeutic purposes, especially with the advent of intravenous immunoglobulin (IVIG) therapy for an increasing number of autoimmune diseases. Examples include, without exclusion, immune thrombocytopenic purpura, Kawasaki disease, Guillain-Barre syndrome, polymyositis/dermatomyositis, vasculitis and systemic lupus erythematosus (SLE). See, Yaniv et al. (September 2000) “Intravenous Immunoglobulin (IVIG) in Autoimmune Diseases Expanding Indications and Increasing Specificity” Research Report of the American Autoimmune Related Diseases Association, Inc. IVIG therapy has also been shown to be effective in the treatment of Alzheimer's disease. Studies have demonstrated anti-Aβ autoantibodies' role in facilitating the clearance of neurotoxic Aβ assemblies and decreasing the serum Aβ in CSF (cerebrospinal fluid), which in turn reduce cerebral Aβ peptide deposition and cognitive decline. See, Szabo et al. (2008) Autoimmuity Reviews 7: 415-420.
Results from an autoantibody assay, however, are only as useful as the quality of the data it produces. Conventional immunodetection methods such as the Farr assay, Crithidia IFA (immunofluorescent assays) and ELISA compromise between specificity and sensitivity and have their respective constraints. See, Hughes et al. (2006) CLI 18(7):12-17. Farr assays, for instance, precipitate immune complexes of dsDNA/anti-dsDNA Abs at high salt concentrations in ammonium sulphate, which causes low avidity dsDNA/anti-dsDNA antibody complexes to dissociate and thereby limits detection to autoantibodies with relatively high avidity. This result is a setback as autoantibodies are highly heterogeneous with respect to their avidity and those of moderate to low avidity may also have clinical significance, as seen in Alzheimer's disease (AD) where IgG autoantibodies bind to epitope on beta amyloid (Aβ) monomers and aggregate with moderate avidity. See, Szabo et al. (2010) Journal of Neuroimmunology 227: 167-174. Furthermore, Farr assays employ a radiolabel and are both labor-intensive and expensive. IFA Crithidia, while capable of detecting autoantibodies of moderate to high avidity, is fairly time-consuming and subjective due to its dependence on slide scoring. ELISA assays are generally more sensitive and susceptible to automation and, thus, often the assay of choice.
Standard ELISA methods are not without shortcomings. They are generally less specific than other methods such as immunopreciptation and immunoelectrophoresis and suffer from a host of issues as documented in the literature. As a case in point, standard ELISA methods have yielded widely disparate estimates on the relative titers of anti-Aβ autoantibodies in AD patients versus aged normal controls. Initial studies of intact plasma specimens by standard ELISA methods ascribed lower titers of endogenous antibodies against Aβ monomers to AD patients than aged-matched non-demented controls (Weksler et al., Exp Gerontol., 37:943-948 (2002)). Subsequent studies reported equal or increased titers of circulating anti-Aβ monomers antibodies in AD patients (Mruthinti et al., Neurobiol Aging, 25:1023-1032 (2004)) based on ELISAs performed on plasma immunoglobulin eluted from Protein G columns at low pH (Akerström et al., J Biol. Chem., 261:10240-10247 (1986)). The higher titers obtained by this method were hypothesized to reflect the presence of a pool of bound anti-amyloid antibodies that were undetected in assays of whole plasma and measurable when freed from antigen by acidification in the course of protein G purification.
Misfolding and aggregation of the Aβ is central to the pathogenesis of AD. The human immunoglobulin G (IgG) repertoire contains autoantibodies against the Aβ peptide that arise in the absence of vaccination or passive immunization and such anti-Aβ autoantibody activity has been detected in the blood of normal adults of various ages and patients with AD (Weksler et al., Exp Gerontol., 37:943-948 (2002); Hyman et al., Ann Neurol., 49:808-810.5 (2001); Mruthinti et al., Neurobiol Aging., 25:1023-1032 (2004); Nath et al., Neuromolecular Med., 3:29-39 (2003); Sohn et al., Frontiers in Bioscience., 14:3879-3883 (2009)). The interest in such anti-amyloid-β autoantibodies has intensified with the discovery that human IVIG containing elevated levels of the autoantibodies have therapeutic effect in AD patients (Dodel et al., J Neurol Neurosurg Psychiatry., 75:1472-1474 (2004); Hyman et al., Ann Neurol., 49:808-810.5 (2001); Mruthinti et al., Neurobiol Aging., 25:1023-1032 (2004)).
Despite recent advances in Alzheimer's research, efforts to accurately measure anti-amyloid-β autoantibodies have been undermined by many obstacles. One obstacle is the existence of multiple classes of human antibodies that recognize linear as well as conformational neo-epitopes on aggregated forms of Aβ. Reports to date have identified endogenous human antibodies against Aβ fibrils (O'Nuallain et al., J. Immunol., 176:7071-7078 (2006)), Aβ oligomers (Moir et al., J Biol. Chem., 280:17458-17463 (2005); Relkin et al., Alzheimer's and Dementia, 3:S196, X (2008); O'Nuallain et al., Biochemistry, 47:12254-12256 (2008)) and conformational epitopes on Aβ monomers (Baumketner A et al., Prot Sci., 15:420-428 (2006)). Other types of amyloid binding antibodies and even catalytic antibodies against Aβ have been reported (Taguchi H et al., J Biol. Chem., 284:4714-4722 (2008)).
The measurement of low-avidity, polyreactive autoantibodies is a particular challenge due to high background binding to empty ELISA wells and assay interference from other plasma proteins and components in the blood samples. See, Szabo et al. (2010) Journal of Neuroimmunology 227:167-174. These problems must be overcome as a large proportion of IgG autoantibodies in IVIG are polyreactive and low-avidity.
Efforts to improve the sensitivity and signal-to-noise ratio of anti-Aβ autoantibody assays have been made, including a radio-immunoprecipitation assay developed by the Brettschneider research team. See, Brettschneider et al. (2005) Biol. Psychiatry 57: 813-816. In another study, pre-assay passage of IVIG over polystyrene and/or agarose columns was done in the hopes of depleting non-specific autoantibody binders. Results were less than ideal as the depletion was not specific to autoantibodies which bind blank plates and depletion reduced the already low concentrations of anti-Aβ autoantibodies, which further complicated measurement. The low-avidity of polyreactive autoantibodies makes them difficult to detect, and the resulting signal intensity is further affected by interference from other plasma proteins and components, leading to underestimates of the anti-amyloid activity.
A common practice to compensate for low autoantibody concentrations, which are common for polyreactive, low-avidity anti-amyloid-β autoantibodies, employs less diluted samples for assay. This technique, however, increases both signal strength and noise. Another approach involves treatment of bound autoantibody-antigen complexes with chaotropic salt (ammonium thiocyanate). See, Szabo et al. (2010) Journal of neuroimmunology 227:167-174.
At the other end of the spectrum, recent methods directed at improving signal strength of the low-avidity, polyreactive autoantibody detection have resulted in over-estimates of anti-amyloid activity. For example, a technique that had shown promise involves the isolation of IgG from human plasma via protein G chromatography and acid elution; the assays resulted in a 50-fold increase in apparent anti-Aβ antibody titers (Li et al., BMC Neurosci., 8:22 (2007)). The high titers were, however, attributed to partial denaturation and artificially induced polyvalency of antibodies in the sample, as evidenced by a 100-fold increase in blank plate binding.
To explain, polyreactive antibodies to both foreign and self-antigens are part of the natural antibody repertoire of humans (Djoumerska et al., Scand J of Immunol., 61: 357-363 (2005)). Most polyreactive antibodies have germ line hypervariable regions, belong to the IgG isotype, display lower affinity and avidity for their antigens as compared to monovalent affinity-maturated antibodies, and have more flexible antigen-binding sites. They are thought to serve as a defense mechanism against pathogens.
The accurate measurement of autoantibodies is key to advancing research in the pathogenesis of Alzheimer's disease and other autoimmune diseases. Likewise, clinical assessment and treatment of patients suspected of or diagnosed with autoimmune disease demand improved assays for autoantibodies. With the discovered potential of IVIG as an Alzheimer's treatment comes an intensified need to overcome some of the impediments to anti-amyloid autoantibody measurement and permit the development of a standard for therapeutic use and further studies. Reliable assays for low-avidity, polyreactive autoantibodies have largely proven elusive. As such, there is a need to develop improved assays for low-avidity, polyreactive anti-amyloid autoantibodies with both sensitivity and specificity. The present invention provides autoantibody assays at low conductivity conditions which increase signal strength without negatively impacting binding selectivity.